TRH has the structure:

The nomenclature of Schechter and Berger (27) is used to describe the positions of the peptide substrate residues (P) relative to the scissile P1—P1′ bond and the corresponding subsets (S) in the active site of the enzyme. In other literature, the right portion of the molecule is called the “prolineamide” or “C-terminal” portion; the centre portion of the molecule is called the “histidyl” portion; and the left portion of the molecule is called the “pyroglutamyl”, “COOH-terminal” or “N-terminal” portion.
TRH was the first hypothalamic regulatory hormone to be characterised and as such plays a central role in regulating the pituitary-thyroid axis. In addition, TRH displays a broad spectrum of stimulatory CNS actions that are independent of the hypothalamic-pituitary-thyroid axis (5-7) and it is also now believed to function as a neurotransmitter and/or neuromodulator within the central nervous system (CNS) (5,6). Based on its CNS-mediated effects, TRH has been shown to have potential use in the treatment of brain and spinal injury and certain CNS disorders including cognitive deficits, epilepsy, and shock (7,8). The mechanisms by which TRH improves these clinical conditions are not yet clear but appear to be mediated, in part, by various other neurotransmitter systems (7,8). The full extent of the potential clinical usefulness of TRH cannot be realised until its functions in the CNS are entirely understood.
Unfortunately, the therapeutic efficacy of TRH is limited by its susceptibility to enzymatic degradation (8). Current evidence strongly indicates that TRH-DE is the principal enzyme responsible for the degradation of neuronally released TRH (9, 10-12). TRH-DE is located on synaptosomal membranes in the central nervous system (CNS) (13,14) and thus, it is strategically placed to play a significant role in controlling TRH signals within the CNS, much like that of acetylcholinesterase in regulating the neurotransmitter actions of acetylcholine. TRH-DE is the only ectoenzyme known to degrade TRH (15) and has been shown to exhibit a remarkably high specificity for TRH. Thus, TRH-DE appears to be an exceptional example of a neuropeptide-specific peptidase (16).
In general, potent and selective enzyme inhibitors are required for establishing the exact role of a particular enzyme and are also powerful tools for investigating the biological functions of the enzyme's substrate. In addition to providing valuable insights into the functional roles of enzymes and their substrates, enzyme inhibitors may be used therapeutically to enhance the clinical effects of an enzyme's substrate either by (a) potentiating the endogenous levels of the substrate and/or by (b) protecting endogenously administered substrate from degradation.
Thus, compounds that potently and selectively inhibit TRH-DE could be used to determine the role of TRH-DE in regulating TRH signals. Because TRH-DE displays a strict specificity for TRH, administration of TRH-DE inhibitors should only affect TRH'S neurotransmitter and/or neuromodulator actions. Therefore, TRH-DE inhibitors would also be particularly attractive for investigating the actions of TRH in the CNS. Furthermore, this exclusivity may offer a therapeutic advantage in cases where TRH-DE inhibitors are used to potentiate TRH's CNS actions. The design of TRH-DE inhibitors, however, is made difficult by TRH-DE's restricted specificity and by the lack of a 3D-structure for TRH-DE.
U.S. Pat. No. 4,608,365 Engel describes a treatment for the amelioration of symptoms of amyotrophic lateral sclerosis and other conditions which result from dysfunction of lower or upper motor neurons by the administration of doses of thyrotropin-releasing hormone by intravenous infusion or subcutaneous injection.
Specificity studies thus far indicate that TRH-DE substrates require a five-membered pyrrolidinone, thiazolidinone or butyrolactone ring in the P1 position (4,17). In addition, several studies indicate that TRH-DE specificity is restricted to tri- or tetra-peptides containing the sequence Glp-His and that the presence of a histidine residue in the P1′ position is essential for TRH-DE activity (2,4,18-20). Recently, the inventor was the first to publish the observation that the naturally occurring TRH-like peptide Glp-Phe-ProNH2 is also a substrate for TRH-DE whereas Glp-Glu-ProNH2 is not (21). This finding has since been confirmed by Gallagher et al. (22). To date, no reports of other TRH-like peptides, with the general structure Glp-X-ProNH2, acting as substrates or inhibitors of TRH-DE have been published.
Some tolerance in the P2′ position is suggested by the observations that both pyroglutamyl-histidyl-prolyl-β-naphthylamnide (Glp-His-ProβNA) and pyroglutamyl-histidyl-prolylamido-4-methyl coumarin (Glp-His-ProAMC) are substrates for TRH-DE (4, 20, 22, 23). Glp-His-Trp is hydrolysed by TRH-DE (18) and substitution of the Pro residue in Glp-His-ProβNA by Ala or Trp does not appear to reduce turnover (4).
TRH and the C-terminally amidated peptides Glp-His-Pro-GlyNE2 and Glp-His-GlyNH2, have all been found to have lower Ki values than their corresponding acids when examined as competitive substrates (24, 19, 22) leading to the suggestion that TRH-DE prefers an amide group at the carboxyl-terminus of peptide substrates (22). In addition, because the Km value observed for Glp-His-ProAMC was approximately ten times less than that for Glp-His-ProNH2 Gallagher et al. (22) have proposed that TRH-DE has a preference for large hydrophobic groups at the carboxyl-terminus of substrates. On the contrary, using compounds present in this application, the inventor has now discovered that the addition of a large hydrophobic group to the C-terminus of TRH and TRH-like peptides causes a reduction in both the catalytic rate of hydrolysis and the specificity constant and that this is a useful feature to incorporate into an inhibitor not a substrate.
Only a few inhibitors have been synthesised that exhibit a significant effect on TRH-DE activity and none of these have been shown to be sufficiently effective for pharmacological studies in vivo (11,25). The most potent of these is N-[1(R,S)-carboxy-2-phenylethyl]-N-imidazole benzyl-histidyl β-naphthylamide (CPHNA) (26). Because CPHNA is not a TRH-DE substrate analogue, the specificity of the interactions of CPHNA with TRH-DE has been questioned (11). Nevertheless, CPHNA appears to reversibly inhibit TRH-DE with an inhibition constant (Ki) of 8 μM and to increase the recovery of TRH released from rat brain slices (26). These results indicate that TRH-DE inhibitors can be used to increase local TRH concentrations and that it may be possible to modulate TRH function in vivo via inhibition of TRH-DE activity.
U.S. Pat. No. 4,906,614 Giertz et al. describes a method of preventing or treating posttraumatic nervous injuries by administering a compound of the formula:
wherein R1 is hydrogen, a lower alkyl group, cyclohexyl or benzyl; Z is one of the groups
if Z is a group (a), R2 and R3 together represent an additional bond between the carbon atoms bearing them, or if Z represents a group (b), R3 is hydrogen; R4 is hydrogen or lower alkyl; R5 is hydrogen, lower alkyl or phenyl, R6 is hydrogen or methyl.
U.S. Pat. No. 5,244,884 Spatola et al. describes thionated analogues of thyrotropin releasing hormone, having the formula:
wherein,
Q, W, X and Y, same or different, are oxygen or sulphur, with the proviso that at least one of Q, W, X and Y is always sulphur;
Z is lower alkyl or (4-imidazolyl)methyl; and the pharmaceutically acceptable salts thereof. The disclosed compounds are stated to highly and selectively bind to TRH binding sites in animal tissues, and their utility in treating a variety of diverse physical conditions is disclosed.
U.S. Pat. No. 5,686,420 Faden describes a series of novel thyrotropin-releasing hormone analogs wherein the C-terminal prolineamide moiety has been preserved, the N-terminal moiety comprises one of five different ring structures and the histidyl moiety is substituted with CF3, NO2 or a halogen. A method of use of the analog for the treatment of neurologic disorders in also provided.
The contents of each of the above-mentioned U.S. Patents is incorporated herein by reference.
The present invention relates to compounds that competitively inhibit TRH-DE and display greater apparent binding affinities (i.e. lower Ki values) for TRH-DE than the endogenous substrate, TRH. These compounds have not been reported to occur naturally. Searches carried out after the priority date of this application have revealed that one of the compounds has been named in published papers.
Burt D. R et al., Brain Research, 93 (1975) 309-328 mention pGlu-Asn-ProNH2 as one of 25 analogues of TRH used in tests of inhibition of TRH binding in the cerebral cortex and pituitary.
Bissette G. et al., Neuropharmacology (1978) 17 (45), 229-37 list pGlu-Asn-Pro-NH2 (Abbott 43689) as a TRH analogue used in tests of analeptic effect in mice.
Mazurov A. A. et al., Russian Chemical Bulletin (1998) 47 (10) 19601964 describe the preparation of (inter alia) Glp-Asn-ProNH2 and the measurement of the antidepressant activity of the compound in rats.
Mazurov A. A. et al., Int. J. Peptide Proteins Res. (1993) 42, 14-19 describe the synthesis of Glp-Asn-ProNH2.
Oliver C. et al., Biochem Biophys. Res. Commun. (1978) 84 (4) 1097-1102 refer to pGlu-Asn-Pro-NH2 as one of 30 TRH analogues subjected to enzymic degradation by rat serum or brain homogenate.
None of these papers discloses or suggests the use of Glp-Asn-ProNH2 as an inhibitor of activity of TRH-DE and there is nothing in their teaching to indicate the line of research leading to the present invention.
All of the other compounds of the invention are believed to be novel.